Our studies reveal overexpression of non-muscle caldesmon (/-CAD), myosin isoform SM-A, and Rho-kinase in the hypertrophied smooth muscle from partially obstructed bladders which present with altered contractility. Furthermore, it exhibits a change from phasic to tonic-type. Neither the relationship between these changes in the isoforms of myosin and CaD, nor their specific effects on detrusor contractility are understood. We propose to test the following hypotheses: 1. The overexpression of 1-CaD in the decompensated bladder is a compensatory change required by the detrusor, which has been changed from phasic to tonic and overexpresses SM-A isoform. We will test this hypothesis by (a) analyzing the expression of CaD isoforms in the detrusors of non-obstructed and partially obstructed SM-B knockout mice and (b) determining if the l- and h-CaD are co-localized with actin filaments in the detrusor myocytes in normal and obstructed SM-B knockout mice by immunofluorescence, confocal, and electron microscopy. These data will tell us whether outlet resistance, in addition to a change in the myosin isoform from SM-B to SM-A, is required to induce a change in the l-CaD expression. 2. The myosin phosphorylation-dependant regulation in decompensated bladder smooth muscle is modulated by ROK-mediated and/or CPI-17-mediated calcium sensitization of force generation. We will test this hypothesis using detrusor tissues from normal, obstructed, and reversed bladders from rabbits, mice (wild-type and SM-B knockout), and humans with increased outlet resistance by (a) determining the expression of ROK by RT-PCR, real-time PCR, and proteomics analyses, (b) analyzing the effect of ROK inhibitor Y-27632 on force maintenance and MLC20 phosphorylation in the intact muscle strips and (c) determining whether CPI- 17, a second signaling pathway dominant in tonic smooth muscle is overexpressed in the detrusor from decompensated rabbit bladders which exhibit tonic-like contractile characteristics. Basic information regarding the ROK-mediated and thin filament-mediated regulation of actin-myosin interaction and force development would help to target molecular steps for the development of therapeutic agents for lower urinary tract diseases associated with smooth muscle contractile dysfunction.